Mpox virus infects and injures human kidney organoids, but responding to antiviral treatment

Mpox/monkeypox virus (MPXV) belongs to the Orthopoxvirus genus of the Poxviridae family. It has a large, linear, double-stranded DNA genome. Since the ﬁ rst identi ﬁ cation in 1970, MPXV is generally con ﬁ ned to tropical regions within African countries. Alarmingly, multi-national mpox outbreaks occurred in 2022 across a large number of non-endemic countries particularly in Europe and America 1,2 . Skin lesions are the most com-mon symptom

switched to Advanced RPMI1640 medium supplemented with 200ng/mL recombinant human FGF9, 1 μg/mL heparin sodium salt (Sigma Aldrich, USA) and 10ng/mL recombinant Activin A (R&D Systems, USA) for one day. Cells were then dissociated by TrypLE Select, and 5×10 5 cells were pelleted by centrifuging at 300 g for 3 minutes in 96-Well Polystyrene Conical Bottom MicroWell™ Plates (Nunc, USA). Then the cell pellets were cultured in advanced RPMI 1640 medium supplemented with 200 ng/mL FGF9, 1 μg/mL heparin sodium salt and 3 μM CHIR99021.
After two days submerged culture, the self-aggregated organoids were transferred on to Polyester Membrane (0.4 μm pore size) Insert (CellQART, DE). After 24 hours treatment with 200 ng/mL FGF9, 1 μg/mL heparin sodium salt and 3 μM CHIR99021, CHIR99021 was removed and organoids were cultured for another 4 days. All growth-factors were then removed, and the organoids were cultured on advanced RPMI 1640 medium for up to 10 days. Virus infections were performed 7 days before the end of the protocol.

In vitro infection and antiviral treatment
Each kidney organoid was inoculated with approximately 5x10 4 PFU MPXV and incubated at 37 o C for 1 hour. After incubation, viral inoculum was removed and organoids were washed with PBS three times. Kidney organoids were then cultured on transwell membrane and medium was supplemented in the lower compartment of transwell plate. Culture medium and organoids were separately lysed (MagNA Pure 96 External Lysis Buffer, Roche, Germany) at 1 hour, 48 hours, 96 hours and 7 days post-inoculation for further analysis. For antiviral drug treatment, after virus inoculation and three times of PBS wash, culture medium was supplemented with tecovirimat (Selleckchem, USA) in serial concentrations, and medium was refreshed at 48 hours and 96 hours.
These kidney organoids were differentiated from two iPSCs lines which were generated from two independent donors. For experimentation, a single organoid was used for each well as an independent replicate. For each experimental condition, individual organoids from different donors were pooled for analysis.

Plaque assay
Infected kidney organoids were stored in 1 ml medium, and centrifuged after 3 times freezing and thawing to collect clear cell lysates before performing plaque assay. Supernatants from infected kidney organoids were directly used for inoculation. Confluent Vero cells in 12-well plates were washed once with PBS. Next, Vero cells were overlaid with 1.2% avicel in advanced DMEM/F12 medium (with 1xGlutaMAX, 1 M Hepes and 1% penicillin/streptomycin) containing ten-fold serial dilutions of samples. Plates were incubated 3-4 days and then fixed by 4% paraformaldehyde (PFA) and stained with 0.1% crystal violet. Plaques were quantified as PFU/mL.

DNA extraction and qPCR
Total DNA was purified from infected organoids or supernatants using Macherey-Nagel

Quantification of MPXV genome copy numbers
Purified MPXV viruses were used to isolate DNA and served as a template for quantifying genome copy number. Primers used to detect MPXV are: (Forward-GGCTCTTCTATCAACCACA; Reverse-AGTCATTATCTCCTCCTCCA). Ten-fold serial dilutions of viral DNA from 10 0 to 10 −7 were prepared and were then quantified by qRT-PCR to generate a standard curve. A standard curve was generated by plotting the log copy number versus the cycle threshold (CT) value ( Figure S1).

Immunohistochemistry and immunofluorescence staining
Kidney organoids were fixed in 4% PFA at 4 o C overnight. Then organoids were embedded in 1% Agarose LE (Roche, Germany) and placed in a cassette for paraffin-embedded specimens.

Genome-wide RNA sequencing and data analysis
For RNA-seq sample preparation, per kidney organoid was inoculated with 5x10 4 PFU MPXV and